The use of arrays for detection and identification of biological and chemical substances is known. An example is fixed nucleic acid hybridization arrays that contain a multiplicity of different hybridization probes immobilized at separate known locations on a planar solid surface, for example a silicon chip or glass slide. At each location in the array, many copies of the same hybridization probe are immobilized. Planar arrays can contain hundreds or even thousands of different areas, each with immobilized hybridization probes. When an analyte containing nucleic acids is added to the array, different nucleic acid sequences will hybridize to probes in different locations. Detection of hybridization at a particular location indicates the presence of a nucleic acid sequence in the sample being analyzed that is complementary to the sequence of the probe immobilized at that location. In general, arrays of this type are prepared in one of two ways: either probes are synthesized in place, as by photolithography techniques, or probes are pre-synthesized and then immobilized. Detection of hybridization is accomplished in any of several ways, including attaching detection labels to analyte substances prior to the hybridization step, addition of an intercalating dye that fluoresces when in contact with double-stranded nucleic acid, adding color-forming moieties to analyte substances for use in a subsequent color-generating step, or by performing sandwich hybridizations in which the targets are bound to capture probes on the array and labeled detector probes are hybridized to the bound targets. It has been found that surface microarrays are subject to substantial variation. Lee, M.-L. T. et al. (2000), Importance of Replication in Microarray Gene Expression Studies: Statistical Methods and Evidence from Repetitive cDNA Hybridizations, Proc. Natl. Acad. Sci. USA 97: 9834-9839.
Another type of array utilizes microcarriers, for example microbeads, having immobilized on their surfaces capture substances, such as hybridization probes for nucleic acids. Individual beads are coated with one or another hybridization probe, and analyte is contacted with a mixture containing at least one bead, generally multiple beads, containing each probe sequence. The beads can be slurried with analyte, which overcomes some of the variation encountered with planar arrays, and then laid down in a planar array of microbeads. Alternatively, the microbeads can be laid down into a planar array prior to contact with analyte. Microbeads may be contacted with analyte, washed and settled into a microscope slide as a planar array. The planar array may also be a pair of narrowly separated transparent plates which trap microbeads in a single layer and are part of a flow cell through which analyte and wash solutions may be flowed. Brenner, S. et al. (2000), Gene Expression Analysis by Massively Parallel Signature Sequencing (MPSS) on Microbead Arrays, Nat. Biotechnol. 18: 630-634. Microbeads can also be settled into depressions etched in the ends of individual strands of a fiber-optic cable bundle containing a large number of strands. Ferguson, J. A. et al. (1996), A Fiber-Optic DNA Biosensor Microarray for the Analysis of Gene Expression, Nat. Biotechnol. 14: 1681-1684. In such distributed arrays, where location does not identify which probe is present, coding schemes are required so that beads containing particular probes can be identified. Rather than laying beads down in a planar array, flow cytometry can be used to interrogate individual beads using, for example, a fluorescence-activated cell sorter. Here again, coding schemes are required.
Simply labeling beads with differently colored fluorophores does not provide a sufficient code, because fewer than ten, practically only seven or eight, different fluorophores can be distinguished. Several complex bead-coding methods have been devised. One coding scheme has been to imbed combinations of fluorophores in the beads. Combinations of colors and intensity comprise a code. Spiro, A. et al. (2000), A Bead-Based Method for Multiplexed Identification and Quantitation of DNA Sequences Using Flow Cytometry, Appl. Environ. Microbiol. 66: 4258-4265. Multiple stimulations and readings of laser light at different wavelengths interrogate each bead to read its code. This scheme requires substantial equipment, cannot deconvolve spectra in real time, and has been limited to about one hundred differently coded beads. Another coding scheme is to imbed tiny stripes of different metals into microcarriers and read the metallic pattern in each microcarrier. This scheme involves complicated microcarrier manufacturing. Another scheme is to embed in each microcarrier a miniature transponder that is energizable by a photocell and transmits a fifty-digit binary code for identification. Such microcarriers can be flowed by a station to detect hybridization and a separate station to decode the microcarrier. A drawback with this transponder scheme is that the microcarriers are not true spheres and fluorescence is to some extent a function of a bead's orientation as it passes a detection station.
Immobilized oligonucleotides have been used for coding purposes. One such system utilizes sixteen different oligonucleotides for coding. Coding is accomplished by deciding in advance, for each of the sixteen types of coding oligonucleotides, whether or not that particular type of coding oligonucleotide will be immobilized on the surface of a given type of bead. Since there are sixteen choices to be made, one for each coding oligonucleotide, the number of different codes (and thus the number of different types of beads) is 2 raised to the power 16, which is 65,536. Beads in a mixture are first fixed to the ends of fiber optic strands in a bundle. The remote ends of the strands are attached to a detector in a fixed pattern. Through a series of sixteen different hybridization reactions with labeled probes, each bead is interrogated and the identity of each bead associated with its fiber optic strand is determined. The bundle can then be contacted with analyte. Oliphant et al. (2002), BeadAway™ Technology: Enabling an Accurate, Cost-Effective Approach to High-Throughput Genotyping, BioTechniques 32: S56-S61. Schemes relying on a serial hybridization are complicated, cumbersome and expensive.
An aspect of this invention is a bead-coding scheme that combines detectable fluorescent labels with a physically induced change in fluorescence to permit coding and optical decoding of hundreds and even thousands of different beads.
Another aspect of this invention is hybridization array methods and reagents, including particularly distributed array methods and reagents, utilizing such a combination coding scheme.
Another aspect of this invention is the use of a coded microcarrrier having immobilized on its surface a plurality of quenched, labeled signaling hairpin molecules each comprising an interacting affinity pair separated by a linking moiety, one member of the affinity pair having bound thereto at least one fluorophore and the other member of the affinity pair having bound thereto at least one quencher, wherein interaction of the affinity pair of each hairpin molecule is disruptable by a physical or chemical change in a condition of its environment, wherein the disruption of the interaction of at least one affinity pair occurs at a first level of said condition and the disruption of at least another affinity pair occurs at a second level of said condition, and where said disruptions are optically differentiable.
Another aspect of this invention is the use in a distributed microarray of a mixture of a plurality of coded microcarriers according to the preceding paragraph, wherein the individual microcarriers each have immobilized thereon a capture probe and wherein the coding scheme for identifying individual microcarriers in said mixture comprises a combination of from three to eight spectrally differentiable fluorophores and at least three affinity pairs disruptable at detectably different levels of said condition, optionally where there is a plurality of identically coded microcarriers having immobilized thereon the same capture probe, and optionally wherein the capture probes are molecular beacon probes.